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Anti-HER2 glycoantibodies and uses thereof

a glycoantibody and anti-her2 technology, applied in the field of therapeutic monoclonal antibodies, can solve the problems of reducing the life span of the circulatory system of the drug, affecting the effector function of the therapeutic antibody, and provoking immunogenicity, so as to improve the therapeutic value, increase the binding affinity of the fc receptor, and increase the activity of the adcc.

Active Publication Date: 2018-06-26
ACAD SINIC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008]Accordingly, one aspect of the present disclosure relates to a composition of anti-HER2 glycoantibodies comprising a homogeneous population of anti-HER2 IgG molecules having the same N-glycan on each of Fc. The anti-HER2 glycoantibodies of the invention can be produced from anti-HER2 monoclonal antibodies by Fc glycoengineering. Importantly, the anti-HER2 glycoantibodies have improved therapeutic values with increased ADCC activity or increased Fc receptor binding affinity compared to the corresponding monoclonal antibodies that have not been glycoengineered.
[0013]In some embodiments, the anti-HER2 glycoantibodies described herein are characterized in that the glycoantibodies exhibit enhanced binding to FcγRIIIA as compared to Trastuzumab.

Problems solved by technology

However, these expression systems suffer from a number of drawbacks that can negatively affect the effector function of therapeutic antibodies.
Their glycosylation machinery often adds undesired carbohydrate determinants which may alter protein folding, induce immunogenicity, and reduce circulatory life span of the drug.
Notably, sialic acid as N-acetylneuraminic acid is not efficiently added in most mammalian cells and the 6-linkage is missing in these cells.
Engineering cells with the various enzymatic activities required for sialic acid transfer has not yet succeeded in providing a human-like pattern of glycoforms to protein drugs.
Each of anti-HER2 IgG molecules in the heterogeneous mixture may not all have the same property, and certain N-linked oligosaccharides bound to therapeutic proteins may trigger undesired effects in patients thus deeming them a safety concern.

Method used

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  • Anti-HER2 glycoantibodies and uses thereof
  • Anti-HER2 glycoantibodies and uses thereof
  • Anti-HER2 glycoantibodies and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Generation of Exemplary Anti-HER2 GAbs

Anti-HER2 GAb301

[0235]The complete removal of N-linked glycan at Asn297 from Fc region of Trastuzumab (Herceptin) is achieved by means of PNGase F, and evaluated with 4-12% Bis-Tris NeuPAGE and LC-MS / MS analysis of tryptic glycopeptides from modified and unmodified IgG. The molecular weights of tryptic glycopeptides helps to determine the potential site of N-linked glycosylation at each asparagine and to elucidate the species of predominant glycans.

Anti-HER2 GAb201

[0236]A glycoantibody derived from conventional Trastuzumab (Herceptin) bearing a glycan structure of GlcNAc-Fuc in the Fc region was treated with one or more endoglycosidases (Endo S and / or Endo F1, Endo F2, Endo F3), and then treated with an alpha-fucosidase to cleave the core fucose with a high cleavage efficiency and lead to a glycoantibody with a monosaccharide GlcNAc in the Fc region.

[0237]Mixtures of Endo S (10 μg), Endo F1 / Endo F2 / Endo F3 (10 μg), Fucosidase (2 mg) and Trastuzu...

example 2

Characterization of Anti-HER2 GAbs

General

[0261]MS spectrometry analysis of of glycoengineered mAb. Intact molecular weights of glycoengineered antibodies were detected by LC-ESI-MS on a LTQ Orbitrap XL ETD mass spectrometer (Thermo Fisher Scientific, San Jose, Calif.) equipped with ACQUITY UPLC (Waters, Milford, Mass.), using a BEH C4 column (1.0 mm×150 mm, 1.7 □m, Waters, Milford, Mass.). Briefly, the gradient employed was 1% buffer B at 3 min to 40% buffer B at 20 min with a flow rate of 50 uL / min, where buffer A was 0.1% formic acid in H2O and buffer B was 0.1% formic acid in 80% acetonitrile. Full-scan MS condition: mass range m / z 800-4000 with Iontrap. Electrospray voltage was maintained at 4.0 kV and capillary temperature was set at 275° C.

[0262]For the analysis of trypsinized glycopeptides, high resolution and high mass accuracy nanoflow LC-MS / MS experiments were done on a LTQFT Ultra (Linear quadrupole ion trap Fourier transform ion cyclotron resonance) mass spectrometer (Th...

example 3

Anti-HER2 GAb ELISA Binding Assay

General

[0272]SDS-PAGE detection of glycoengineered Herceptin antibodies. All the SDS-PAGE analysis was performed with NuPAGE® Novex® 4-12% Bis-Tris gel (Invitrogen) ether with or without DTT addition.

[0273]Recombinant human HER2 protein (purchased from Sino Biological Inc.) (1 μg / mL) in 100 μL carbonate coating buffer (pH 10) was coated onto 96 well high binding plates at 4° C. for overnight. 2% of bovine serum albumin in PBST was then added to block plates at r.t. for 1 hr and subsequently a series dilution of anti-HER2 GAbs 101, 102, 104, 105, 106, 107, 108, 110 and 111 were added to plates and incubated for one hour. After incubation, HRP conjugated anti-human IgG was added to the reaction mixture and incubated for one hour. An aliquot of OPD substrate was added, and the absorbance at 450 nm was recorded. Throughout the experiment, plates were washed with PBS twice after each incubation step. Results of ELISA binding are shown in Table 8.

[0274]

TAB...

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Abstract

The present disclosure relates to a novel class of anti-HER2 monoclonal antibodies comprising a homogeneous population of anti-HER2 IgG molecules having the same N-glycan on each of Fc. The antibodies of the invention can be produced from anti-HER2 monoclonal antibodies by Fc glycoengineering. Importantly, the antibodies of the invention have improved therapeutic values with increased ADCC activity and increased Fc receptor binding affinity compared to the corresponding monoclonal antibodies that have not been glycoengineered.

Description

RELATED APPLICATIONS[0001]This application claims the benefit of priority to U.S. provisional applications U.S. Ser. No. 62 / 003,104, filed May 27, 2014, U.S. Ser. No. 62 / 020,199, filed Jul. 2, 2014, and U.S. Ser. No. 62 / 110,338, filed Jan. 30, 2015. The contents of each of which is hereby incorporated by reference in its entirety.BACKGROUND OF THE INVENTION[0002]Fc glycosylation has been an important subject in the field of therapeutic monoclonal antibodies. Fc glycosylation can significantly modify Fc effector functions such as Fc receptor binding and complement activation, and thus affect the in vivo safety and efficacy profiles of therapeutic antibodies.[0003]Several expression systems based on genetically engineering have been reported to produce therapeutic monoclonal antibodies. These include yeasts such as Pichia pastoris, insect cell lines, and even bacteria. However, these expression systems suffer from a number of drawbacks that can negatively affect the effector function ...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C07K16/32C07K16/40C12P21/00C07K16/28A61K31/517A61K39/395
CPCC07K16/40A61K39/3955C07K16/283C07K16/32C12P21/005C12Y302/01C12Y302/01051A61K31/517C07K2317/92C07K2317/14C07K2317/24C07K2317/31C07K2317/40C07K2317/41C07K2317/732A61P35/00A61P43/00C07K2317/21C07K2317/72C07K2317/73A61K2039/505A61K2039/507
Inventor WONG, CHI-HUEYWU, CHUNG-YI
Owner ACAD SINIC
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